EP3362092A1

EP3362092A1 - Manufacture and use of personalized hyperimmune egg in psoriasis treatment

Info

Publication number: EP3362092A1
Application number: EP16716713.9A
Authority: EP
Inventors: Chiurciu CONSTANTIN; Chiurciu VIORICA; Lucica SIMA; Iuliana MIHAI; Victor Patrascu Ionel
Original assignee: Romvac Co SA
Current assignee: Romvac Co SA
Priority date: 2015-10-16
Filing date: 2016-01-08
Publication date: 2018-08-22
Also published as: MA42973A; WO2017065626A1; RO130965A8; RO130965A0

Abstract

This invention refers to the discovery of the way of treating psoriasis using personalized chicken immunoglobulin therapy. Chickens are immunized with an immunogen prepared from skin wound samples taken from each and every patient. This invention refers to the a particular immunogen, taken from the patient, mixed with an adjuvant and which, when administered in chickens, yields a specific immune response within an appropriate period of time so that the Ovopach eggs obtained from these chickens should be used under stringent conditions in the psoriasis treatment of the patient. The immunogen is polyspecific and contains microbial and cellular antigens found in the psoriasis wounds of the patient. The specific immune response from the immunized chickens is monitored on daily basis by extraction of immunoglobulins (Ig) from the egg yolk (Y) and qualitative and quantitative assessment by ELISA, radial immunodiffusion and Ouchterlony immunodiffusion assay. The immune response of chickens, expressed by the yolk-extracted antibodies, is a complex, multiple response against all antigenic stimuli present in the pathogenic product taken from the patient.

Description

DESCRIPTION OF INVENTION

Title:

MANUFACTURE AND USE OF PERSONALIZED HYPERIMMUNE EGG (OVOPACH) IN PSORIASIS

TREATMENT

Inventors:

Chiurciu Constantin, Viorica Chiurciu, Lucica Sima, Iuliana Mihai, Patra§cu Ionel Victor

TECHNICAL FIELD

Immunology, medicine, treatments, prevention, biotechnology applications

This invention describes the production of Ovopach hyperimmune egg and proteins extracted from it for the personalized treatment of psoriasis patients. Psoriasis is an autoimmune chronic, sometimes hereditary disease, affecting about 3% of people around the world, usually between the ages of 11 and 45 years. Psoriasis is considered an incurable disease. Treatments known so far have been used to control disease symptoms. The experts purport that the causes of psoriasis are unknown, but the disease is a result of excessive growth and multiplication of keratocytes, the cells in the deep layers of skin. Psoriasis is considered an autoimmune disease. In this case, T lymphocytes accidentally recognize the normal epidermal cells as being foreign antigens.

From our experience up to this date, the use of a personalized immunoglobulin or mixture of personalized proteins extracted from the Ovopach egg can stand as a chance for each and every patient who suffers from psoriasis with wounds infected with specific pathogenic germs which are sensitive to or resistant against all antibiotics. It is known that in most chronic illnesses the impaired tissues have a reduced natural resistance and the modified and immunologically deficient cells allow the development of a microbial flora of varying nature and pathogenicity (1).

The presence in this case of specific multiple anti-pathogens antibodies, modified opportunistic flora, cells different from normal from structural and antigenic point of view, reduces the efficacy of antibiotic treatment (frequent relapse and resistance occurrence). These data enabled us to prepare and administer polyvalent specific chicken immunoglobulins (IgY-specific).

In this treatment program we used the method described by Jerne (20) which refers to the preparation of an Abl idiotypic antibody which, when orally administered and absorbed in the bloodstream, would be treated as antigen by the naive T lymphocytes and its antigen information should be copied as negative image. It puts this information in its own DNA and a clone is immediately formed, bearing this immunological message. T lymphocytes activate the B lymphocytes transferring to them the negative image as negative information to the T lymphocyte negative and which, at the patterning of IgG antibodies, becomes positive image identical with the information on the IgY-idiotypic orally administered in people and prepared from chickens.

This invention refers to the preparation of the personalized hyperimmune egg Ovopach against the antigens prepared from pathogenic agents, modified opportunistic flora, cells different from normal from structural and antigenic point of view present in the pathological material sampled from the patient; the following step is that further lab assays should identify pathogenic germs and their antibiotic resistance. The pathological product sampled from the skin wounds of patients can be considered a total immunological cell-microbial stimulus (1).

The antigen obtained after cultivation of the pathological cell material sampled from the patient in culture medium for 24 or 48 hours at 37 °C, is inactivated in formalin or thermally or by gamma rays. The immunogen is then prepared and administered in chicken. In order to carry out an efficient treatment using immunoglobulins extracted from the yolk (IgY) of chickens immunized with specific antigens mixed with adjuvant QS21, these IgY must be specific, with direct effect against the pathogenic germs in the organism of the patient. Scientifically, the Ovopach hyperimmune egg contains anti-anti-personalized pathogenic complex (AIAACPI) idiopathic antibodies which recognize the epitopes defined by the peptides in the personalized pathogenic product (15). The inactivated antigenic product mixed with adjuvant QS21 is used for the fast immunization of laying chickens. For this purpose, 24-50 weeks old chickens at high laying season are used. The inoculums is assessed depending on the total protein determined by spectrophotometer or Patrascu radial ID method (unreleased data). The isolation of pathogenic germs found in this pathologic product is carried out at the same time with immunization. Depending on the species of pathogenic germs, resistance or sensitivity to antibiotics, they can be used during treatment with personalized IgY.

The immune response of immunized chickens is daily monitored by highlighting IgY in egg yolk. The specific qualitative and quantitative . immune response against the administered antigen shall be monitored. The immune response of each and every chicken is different from the quantitative point of view. Eggs are collected from chickens producing amounts of IgY higher than 1,500 OD by ELISA. A mixture is performed from yolks of chickens with more than 1,500 OD in the egg by ELISA, from which IgY is immediately extracted by Patrascu method. The extracted IgY is twenty times concentrated by tangential filtration. The IgY concentration is determined by ELISA and diluted to the amount expressed in mg/dose depending on treatment. The personalized IgY is sterilized by filtration through 0,45 μιη and 0,22 μιη Millipore filters.

Much more eggs can be collected in the following days, which can be used as such, freeze- dried as whole egg or immunoglobulin and/or purified proteins.

DESCRIPTION OF THE STAGE OF TECHNIQUE INCLUDING

REFERENCES

The anti idiotypic antibodies were first and independently described by Henry Kunkel and Jacques Oudin (1963). They both noticed that an animal immunized with an antigen generates an antibody response (Abl). If these Abl antibodies are isolated and inoculated in other naive animals, these animals produce anti Abl antibodies. These Ab2 antibodies are called anti- idiotypic antibodies because they recognize an epitope on Abl which is unique for Abl . Oudin noticed that the antigenic specificity of Abl tends to correlate with the idiotypic expression of Abl. In other words, various antibodies were defined as Abl -specific antibodies or a group of Abl antibodies linked to a specific antigen. Later, Alfred Nisonoff and others described two types of Ab2, namely Ab2a and Ab2 β, if Ab2 was capable of binding to Abl in the presence of the antigen. The anti idiotypic antibody Ab2p does not bind to Abl in the excessive presence of the antigen, maybe because Ab2 binds with the binding sites of Abl . However, the anti-idiotypic Ab2a antigens binds with an idiotype distinct from the Abl antigen site.

The idiotypic antibodies were thoroughly described by Niels Jerne from the Pasteur Institute in Paris in 1976 (5), and the phenomenon was further investigated for the purpose of using these immunoglobulin molecules in the treatment of various illnesses, and as a form of new vaccine as well (1-12).

Studies were carried out on anti idiotypic Ab2 antibody, rabies immunoglobulins on dogs, anti gpl20 HIV on cat and chicken (unreleased data).

Idiotipyc IgY anti- matrix T cells -B cell copy - IgG anti-idiotipyic

Figure 1. Mechanism of anti-anti-idiotypic IgG formation

The immunological data transfer is carried out from chicken to human, via IgY (Abl) by the naive T lymphocytes, from T lymphocyte clones to B lymphocytes which take over the negative - negative data and reproduce a positive IgG similar to the original IgY-idiotypic (Patrascu I.V.). The artificial passive immunity is generally produced from the blood which contains large amounts of specific antibodies. Another modern form of artificial passive immunity was obtained by using immunoglobulins (Ig) extracted from the egg yolk (Y) which contains large amounts of specific antibodies. For a better development of this production method and for the maximum improvement of immunological, biochemical and morphofunctional properties of IgY, chickens (Gallus domesticus) are actively immunized with antigenic stimuli prepared for this purpose. The chicken immune response is found in blood and yolk of eggs which are collected after the antibody level can be detected in blood.

The artificial passive immunity provides immediate protection with antibodies against the microorganisms the antibodies have been generated against. These antibodies produced by the actively immunized chicken cannot be produced by the patient because he is not capable of

producing antibodies rapidly, he has no immunocompetence. The person receiving IgY will have a temporary passive immunity for as long as the antibodies last in the organism. This type of immunity acts immediately when the patient needs protection, with antibodies provided by other person or, in this case, by the chicken. As per the information from the CDC Atlanta, Georgia USA, which has studied the immunity of a group/ population, there are people who can receive blood products, such as immunoglobulins which provide immediate protection against a specific disease. This is the utmost benefit of passive immunity because the protection is instant, while the active immunity from vaccines requires time, several weeks maybe, to reach an appropriate protection level (14).

The use of IgY for the prevention and treatment of pathogenic germ infections in humans and animals involved the oral administration of certain drug formulas containing active antibodies. Research to this extent carried out from 1893 until 2015 intended the preventive or curative administration in people and animals of some IgY obtained in the poultry immunized with bacterial or fungal strains, yeasts or reference (distinct) viruses or with bacterial strains isolated directly from patients (12).

There is no public information regarding the preparation of certain chicken immunoglobulins after immunizing them with the pathological material from the skin (sketch no. 2. Patrascu I.V.)

These procedures were for the first time carried out in Romania and are the object of this patent. Specific IgY immunoglobulins were prepared in Romvac laboratories by active immunization of chickens with an antigen obtained by cultivation of the pathological material directly in the culture medium at 37°C for 24-48 hours; the crude antigen obtained was mixed with adjuvant QS21 and administered in chickens at high laying season. Eggs were collected at different periods of time to assess the evolution of the immune response from the yolk of chickens.

The literature states that these eggs, used entirely or for the preparation of specific IgY, are collected starting from the third week after the last vaccination, which is 6 weeks from the first inoculation 6, that is 42 days. Another 2-3 days are required for the use of specific IgY making possible the use of these products in people after 45 days.

Except for the trial conditions, these products are available on the market and can be used right after the recommendation of the physician. These products are used for the prevention or treatment of infections with identified known pathogenic germs for which there is specific IgY. This internationally-accepted methodology cannot be used in patients infected with pathogenic germs for whom the antibiotic treatment had no results or for whom there is no specific IgY.

An egg (Ovopach) was developed under these conditions using a new manufacture and control protocol of specific IgY produced by the chicken and the antigenic stimulus administered together with the adjuvant QS21. The multiple antigenic stimulus is represented by pathogenic agents, modified opportunistic flora, cells different from normal from structural and antigenic point of view (1) sampled from the patients.

REFERENCES

1. C. Gh. Bacanu, §. A. Bacanu. Autovaccin celulomicrobian total complex (AVCMTC).

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2. LF HAAS. Emil Adolph von Behring (1854-1917) and Shibasaburo Kitasato (1852- 1931 ) J Neurol Neurosurg Psychiatry 2001 ;71 :62 doi: 10.1 136/jnnp.71.1.62

3. Fox JP, Elveback L, Scott W, et al. Herd immunity: basic concept and relevance to public health immunization practices. Am J Epidemiol 1971 ; 94: 179-89.

4. Anderson RM, May RM. Vaccination and herd immunity to infectious diseases. Nature 1985; 318:323-9.

5. Fine PEM. Herd immunity: history, theory, practice. Epidemiol Rev 1993; 15:265-302.

6. Fine PEM, Mulholland K. Community immunity. In: Plotkin SA, Orenstein WA, Offit PA eds. Vaccines. 5th ed. Chapter 71. Philadelphia, PA: Elsevier Inc., 2008:1573-92.

7. John TJ, Samuel R. Herd immunity and herd effect: new insights and definitions. Eur J Epidemiol 2000; 16:601-6.

8. Stephens DS. Vaccines for the unvaccinated: protecting the herd. J Inf Dis 2008;

197:643^15.

9. Heymann D, Aylward B. Mass vaccination in public health. In: Heymann D, ed. Control of communicable diseases manual. 19th ed. Washington, DC: American Public Health Association, 2008.

10. Topley WWC, Wilson GS. The spread of bacterial infection: the problem of herd immunity. J Hyg 1923; 21 :243-9

11. Vaccines and Immunisations. Center for Diseases Control and Prevetnion 1600 Clifton Road Atlanta, GA 30329-4027, USA. 19.05.2014

12. Cerere de brevet nr. A/00156 din 25.02.2014 - "Procedeu de ob†inere si utilizare a imunoglobulinelor de gaina (IgY) " Patrascu Ionel Victor, Viorica Chiurciu, Chiurciu Constantin si Georgiana Topilescu

13. Cerere de brevet nr. A/00179 din 05.03.2014 - "Metode de evaluare imunologica a activita ii specifice a imunoglobulinelor de pasare (IgY)" Patrascu Ionel Victor, Viorica Chiurciu, Chiurciu Constantin si Georgiana Topilescu .

14. Cerere de brevet nr. A/00810 din 29.10.2014 - "Producerea si folosirea oului hiperimun PC2" Patrascu Ionel Victor, Viorica Chiurciu, Chiurciu Constantin, Mariana Oporanu, Georgiana Topilescu §i Iulia Mihai.

15. Angel Alberto Justiz Vaillantl , Patrick Eberechi Akpakal *, Norma McFarlane- Anderson2, Monica P. Smikle3 and Wisdom Brian. The Chicken and Egg System for the Development of Anti-Idiotypic Vaccines. Vaccines & Vaccination. Vaillant et al., J Vaccines Vaccin 2012 , 3 : 2

16. Lin-Hui Wang; Xiao-Yu Li; Li-Ji Jin; Jian-Song You; Ye Zhou; Shu-Ying Li; Yong-Ping Xu. Characterization of chicken egg yolk immunoglobulins (IgYs) specific for the most prevalent capsular serotypes of mastitis-causing Staphylococcus aureus. Veterinary microbiology 201 1 ; 149(3 -4) :415 -21

17. Angel Alberto Justiz Vaillantl, Patrick Eberechi Akpaka, Norma McFarlane- Anderson, Monica P. Smikle and Wisdom Brian. The Chicken and Egg System for the Development of Anti-Idiotypic Vaccines. J Vaccines Vaccin 3: 137 doi: 10.4172/2157-7560.

18. Ainsworth A.J., and J E Brown. Detection of in ovo derived idiotypic antibodies. I. A model for maternal-neonatal idiotype network studies.Anna Vangone, Safwat Abdel- Azeim, Ivana Caputo, Daniele Sblattero, Roberto Di Niro, Luigi Cavallo, Romina Oliva. Structural Basis for the Recognition in an Idiotype-Anti-Idiotype Antibody Complex Related to Celiac Disease. PLOS ONE: July 2014, Volume 9 Issue 7 pag. 1-12.

Jerne N K. Towards a network theory of the immune system. Ann Inst Pasteur Immunol 1974. 125C:373-389

Angel A Justiz Vaillann Sehlue Vuma. The Chicken Egg: "A Nature's Miracle". Cell& Developmental Biology. 2015, 4: 1.

Patent WO1994014469 Al Jul 7, 1994. Rodkey L.S., Seferian P.G. Multivalent abl antibodies as vaccines.

Anna Vangone, , Safwat Abdel-Azeim, Ivana Caputo, Daniele Sblattero, Roberto Di Niro, Luigi Cavallo, Romina Oliva. (2014) Structural Basis for the Recognition in an Idiotype-Anti-Idiotype Antibody. PLoS ONE 9(7):

Angel Alberto Justiz Vaillantl, Patrick Eberechi Akpakal *, Norma McFarlane- Anderson2, Monica P. Smikle3 and Wisdom Brian The Chicken and Egg System for the Development of Anti-Idiotypic Vaccines. Journal of Vaccines & Vaccination, April 26, 2012

Shofi qur Rahman, Sa Van Nguyen, Faustino C. Icatlo Jr. Kouji Umeda and Yoshikatsu Kodama. Oral passive immunotherapeutics.A novel solution for prevention and treatment of alimentary tract diseases. Human Vaccines & Immunotherapeutics 9:4, 1-10; April 2013;

A.J. Ainsworth and J.E. Brown Detection of in ovo derived idiotypic antibodies. I. A model for maternal-neonatal idiotype network studies. The Journal of Immunology (Impact Factor: 4.92). 09/1991 ; 147(3):910-4

Alberto Justiz Vaillant, Patrick Eberechi Akpaka, Norma McFarlane-Anderson, Monica P. Smikle and Wisdom Brian. Chicken and Egg System for the Development of Anti- Idiotypic Vaccines. J Vaccines Vaccin 2012, 3: 137

Viorica Chiurciu, I.V. Patrascu, C.Chiruciu, Georgiana Topilescu. Specific Chicken Immunoglobulins (IgY) for Prevention and Treatment and Human Bacterial Infections. European Biotechnology Congress Lecce 2015

Patrascu I.V., Viorica Chiurciu, Chiurciu C, Georgiana Topilescu, Maria Oporanu, lulia Mihai, Georgiana Radu, Andreia Dinu, Badica I., Stoica C, Angelescu C, Alina Gheor, Oltean G. Ovotransferina PC2 (4). O forma noua immunogenic active la nivel molecular. Sesiunea Academiei Romane din 28 februarie 2015

Patrascu I.V., C. Chiurciu. Oul hiperimun. Sesiunea Academiei Romane din 28 februarie 2015

Cerere de brevet nr. A/00156 din 25.02.2014 - "Procedeu de obfinere si utilizare a imunoglobulinelor de gaina (IgY) " Patrascu Ionel Victor, Viorica Chiurciu, Chiurciu Constantin si Georgiana Topilescu

Angel Alberto Justiz Vaillant, Norma McFarlane-Anderson, Patrick Eberechi Akpaka, Monica P Smikle, Niurka Ramirez and Armando Cadiz. Use of Dot Blots Analysis in the Separation of Anti-HIV Antibodies in Animals. J Chromat Separation Techniq 2013, 4:5 Kphler G, Milistein C.Continous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256:495-497

Oudin J., Michel M. Une nouvelle forme d'allotype des globulines gama du serum de lapin, apparemment liee a la specificite anticorp. RC. Acad. Sci. Paris 1963; 255:805-808 Ban, N., Escobar, C, Garcia, R., Hasel, K., Day, J., Greenwood, A., McPherson, A. Crystal structure of an idiotype-anti-idiotype Fab complex. (1994) Proc.Natl.Acad.Sci.USA 91 : 1604-1608 BRIEF DESCRIPTION OF INVENTION

Short description of technical solutions

The objective of this invention is to obtain Ovopach hyperimmune egg as product and method of preparation, active immunological Ovopach specific against bacterial antigens, yeasts, fungi, cell structures present in the pathological material sampled from the psoriasis skin wounds of the patient.

An intensive research program was created for the preparation of specific Ovopach eggs, with the following purpose: obtaining Ovopach with specific effect against various epitopes and application of some control methods which enable the accurate assessment of their effects on the target antigen (9, 10, 14). The monovalent or polyvalent antigen was used in mixture with adjuvant QS21 and an immunomodulator hereinafter referred to as P, as per the methodology described in the invention patent applications submitted to OSIM (12, 13, 14).

By using laboratory techniques like the agar gel immunodiffusion assay, the radial immunodiffusion assay, ELISA identification assay and ELISA specificity assay, the immune response of Ovopach-producing chickens was defined by highlighting and quantification of the immune effectors found in the aqueous extract from the yolks of immunized chickens.

The pathological material used for the rapid immunization of chickens in order to obtain an appropriate specific immune response (1,500 OD or higher by ELISA) is used in vitro and for the isolation, identification and description of various microorganisms existing in this product. This activity, including the antibiogram, contributes to the description of psoriasis wound infections in the patient.

Various products can be prepared from the Ovopach eggs and they can be used in case of emergency for psoriasis treatment in the patient whom the pathogenic material has been collected from. Among these, there are IgY solution to 200 mg/80 ml, IgY solution to 25 mg/ml in 2 ml vials, whole egg, whole freeze-dried egg, IgY ointment to 20 mg/ml, IgY spray to 20 mg/ml which can be locally or generally administered. This invention refers to the preparation and description of the hyperimmune egg called Ovopach intended for psoriasis treatment, the extraction stages of which are described in the Invention Patent (14).

Ovopach hyperimmune egg has a simultaneous specific reaction against one or more antigens used for chicken immunization.

The anti-psoriasis Ovopach hyperimmune egg is obtained from the chickens immunized with the antigen obtained from the pathological material samples from the patient, mixed with adjuvant Q by a single intramuscular administration in four distinct sites in the breast of over 24 weeks old laying chickens. The eggs can be used as such or a whole freeze-dried or atomized egg can be prepared from these eggs and IgY can be extracted after detection in the egg yolk of a specific antibody level of 1500 OD or higher by ELISA. The eggs with appropriate response are used for this purpose.

One of the objectives of this invention is the registration of anti-psoriasis Ovopach as biological product, of its method of preparation and of the method of treatment of psoriasis.

BRIEF DESCRIPTION OF PROCEDURES

This patent describes the biological anti-psoriasis product Ovopach and the method of treatment of psoriasis based on the description below:

To obtain the anti-psoriasis Ovopach hyperimmune egg, chickens are immunized at the age of 24-30 weeks, in the high laying season, with a pathological product sampled from the patient. This product contains specific pathogenic germs which represent a mixture of proteins present after 24-48 hours at 37 °C in the culture medium inactivated in formalin, thermally or by gamma rays, mixed with adjuvant QS21 and administered intramuscularly. The amount of protein mixed with adjuvant QS21 cannot be higher than 200 mg per ml diluted in PBS per dose.

5 - 10 chickens are used, marked by tags applied on both wings, kept in separated cages and the eggs are collected separately from each and every animal.

Each chicken is immunized with 2 ml inoculums, in 4 distinct sites of the pectoral muscles. If 14 days after inoculation there is no specific immune response against the immunization antigen at minimum 1,500 OD by ELISA, the immunization must be carried on according to the program described in the Invention patent application no. A/00810 from 29.10.2014 (14). In emergency cases, an antibody concentration can be performed by tangential filtration.

Combined powder feed must be administered ad libitum throughout the treatment period.

The immune response is assessed directly by monitoring the eggs collected from the immunized chickens. The yolk harvested from each egg is treated with PEG 6000 for IgY extraction. IgY is tested separately identifying the chickens with an immune response above 1,500 OD by ELISA.

The eggs with immune response below 1,500 OD by ELISA can be used for obtaining IgY by concentrating it.

From the mixture of 10 egg whites and 10 yolks of the same eggs, 5 ml of mixture are harvested, extracting ovotransferrin (OTf) and IgY, each by the method specified in the patented technology (12, 13, 14). The yolk extract, called IgY, and the egg white extract, called OTF, are monitored for the immune response by ELISA, radial ID and ID Outcherlony.

14 days after the last immunization, the Ovopach hyperimmune eggs are collected and used for various purposes.

DETAILED DESCRIPTION OF INVENTION

Obtaining anti-psoriasis Ovopach hyperimmune egg and the biological products extracted from it

This biological product and its derivatives are personalized products and the antigens used for the immunization of laying chickens are prepared from the pathological material sampled from a single patient. Ovopach hyperimmune egg and yolk immunoglobulins contain specific antibodies against the pathogenic germs present in the pathological material sampled from the patient, which implicitly contains one of the main triggers of the disease.

The method of preparation of Ovopach and its byproducts involves a self-methodology for the first time in the world.

Preparation of antigen

The personalized antigen is prepared from the pathological material sampled from the psoriasis wounds of the patient. This product contains pathogenic agents, modified opportunistic flora, cells different from normal from structural and antigenic point of view.

According to this invention, the pathogenic material is harvested from each patient and the finished product is personalized and represents the hyperimmune egg or the byproducts. The pathological product found in the transport medium is cultivated on liquid culture media for 24 or 48 hours. The cell material is prepared separately by centrifugation at 5000 rpm at +4 °C. The mixture of these products is inactivated in formalin or thermally or under gamma rays, washed three times in phosphate buffer (PBS) and centrifuged at 4000 rpm for 15 minutes. The sediment is resuspended in PBS at predetermined concentration. 1. Immunization of conventional or SPF laying chickens

The antigen is administered by intramuscular inoculation of 0.5 ml in four distinct sites on the chest muscles of conventional or SPF laying chickens. The antigen is inoculated three times every 14 days. The presence of specific antibodies in blood and eggs is tested after the second administration, by ELISA and ID. Eggs are harvested 14 days after the third antigen inoculation, when the antibody titer is assessed on regular basis from the eggs of chickens immunized with the target antigen (12).

The immunization of laying chickens with a target antigen is a well known technique. This invention can undertake any method of chicken immunization which consists of the administration of the target antigen by any route: subcutaneous, intracutaneous, intramuscular, intravenous.

Adjuvant QS21 was used for this invention. Other types of adjuvants can also be used such as complete or incomplete Freund adjuvant or a mixture of them.

Using antigen QS21 mixed with the target antigen enhances the immune response, induces no local reactions and has been demonstrated to be efficient for the production and maintenance of a high titer for a long period of time.

2. Control of the immune response of laying chickens immunized with personalized antigen

The presence of specific immunoglobulin against the prepared and administered antigen is separately monitored starting from the tenth day after antigen administration. When more than 50% of the immunized chickens transfer in the yolk the right amount of immunoglobulin, 2,500-3,000 OD by ELISA or minimum 1,500 OD by ELISA, the eggs can be collected and used for treatment purposes either as raw, mixed in warm milk or other juices. The specific immunoglobulin can be extracted from the Ovopach hyperimmune egg using the previously patented methodology of preparation and control (12).

METHODS RECOMMENDED FOR THE USE AND DESCRIPTION OF

INVENTION The examples below are for illustration only and have no intention of restricting the purpose of this invention.

Example 1

Preparation of the personalized antigen

The personalized antigen is prepared from the pathological material sampled from the patient. This material can be sampled from the skin surface (scalls). The pathological product transported in vials to the lab is transferred in nutritive broth and incubated for 24-48 hours at 37 °C. The finished product together with the cell debris are inactivated in formalin or thermally or under gamma rays and purified by washing. The sediment is used as antigenic material mixed with adjuvant QS21. Microorganism inactivation control is performed after preparation of the antigenic mass.

Laying chickens immunization

Chickens at high laying season and raised in separated cages are immunized by receiving 2 ml antigen four distinct sites in the pectoral muscles. If the immune response monitored during 10-14 days after the first administration of the antigen is appropriate (2,000-3,000 OD by ELISA), the eggs of these chickens can be used either as administered as such or by extracting immunoglobulins from the yolk. The immunization program can be carried on and the eggs collected from immunized chickens will be used for IgY extraction, purification of egg white and yolk proteins and preparation of a purified protein complex.

The personalized IgY is prepared as per the description in the Patent application no. A/00156 from 25.02.2014 (12) Example 2

Ovopach hyperimmune egg filling

Ovopach hyperimmune egg filling is carried out as per the methodology described in the Patent application no. A/00156 from 25.02.2014 (12).

For optimum use, this personalized biological product is filled as follows:

1. Whole Ovopach egg administered in raw state in warm milk or other liquids;

2. Ovopach egg yolk, atomized or freeze-dried (maximum 5% residual moisture)

3. Immunoglobulin (IgY) extracted from Ovopach egg yolk (200 mg/80 ml liquid, 50 mg/ml, spray, Vaseline, granules, capsules)

4. Purified protein mixture extracted from Ovopach, from the yolk (IgY) and egg white (ovo transferrin, ovomucin, lysozyme).

Example 3

Final control of Ovopach personalized egg a) The below described methods of control are used for the approval of the anti-psoriasis Ovopach personalized egg, the final form of administration in solution for skin, oro- pharyngeal-gastro-intestinal purposes :

- Microbiological sterility;

- Determination of specific Ovopach activity by ELISA, ID, radial ID, depending on the initial composition of the pathological material sampled from the patient.

- Determination of IgY content by ELISA;

- Determination of bacterial growth inhibition activity by IMB-specific PaChi assay b) Only the control-appropriate batches of Ovopach are approved for human consumption.

IgY prepared from the hyperimmune egg Ovopach proved to be capable of specifically reacting with epitopes on the bacteria and fungi in the pathological product sampled from the patient and used for chicken immunization, resembling to that of immunoglobulin (IgY) extracted from the yolk of the same eggs.

Example 4

Ouchterlony agar gel immunodiffusion assay revealing IgY

Principle:

The ID assay is based on the migration of antigen (IgY) and antibodies (anti IgY rabbit serum) to the agar gel where they specifically mix and form a precipitate revealed as a precipitation line.

All controls carried out by agar gel immunodiffusion assay were intended to identify IgY Ovopach against Romvac substandard. These controls belong to the first category of controls used for identification of IgY-Ovopach molecules and for confirmation whether they are immunoglobulins or not.

Each reaction is commented upon under the picture.

ID assay with anti IgY rabbit IgG.

IgG was placed in the central well and in wells no. 1 and 4. Romvac substandard IgY anti S. aureus was placed in wells 2 and 6. IgY Ovopach was placed in wells 3 and 5. It is obvious that the precipitation lines generated by the substandard and IgY Ovopach agglutinate, demonstrating that the products belong to the same protein and antigenic group.

In this control involving the ID assay, the international reference standard was provided by Abeam USA. Anti-avian rabbit IgY was distributed in wells 1, 4 and central. IgY-PC2 ROMVAC substandard was distributed in well 2, IgY-ABCAM-USA was distributed in well 3, IgY-PC2 sediment from PEG precipitate was distributed in well 5, and supernatant from PEG-precipitated Tf-PC2 was distributed in well 6. All IgY samples had positive reaction with anti-avian IgG The precipitation lines between wells 2 and 3 are double and agglutinate. The precipitation lines between wells 5 and 6 agglutinate. This picture obtained by microreaction shows that IgY extracted by PEG precipitation is more concentrated and pushed the reaction line to the central well.

Ovopach radial immunodiffusion identification assay

Anti-IgY IgG is mixed with the agar gel in proportion of 2 ml whole/g of agar. 3 or 6 mm wells are performed in gel. IgY Ovopach is distributed in these wells and it can be from various batches or at various concentrations. The reaction plate is incubated at +22 °C for 20- 24 hours, then it is read under indirect light. The presence of a precipitation plate proves the

occurrence of the antigen/ant o y react on. e eterm nat on of the diameter, deduction of the diameter of the well and the value divided by two represent the migration value of each and every product. When the diameter of the reaction is smaller than that of the control, the product is concentrated. If the Ovopach reaction is higher than that of the control, the product can be diluted. Example 5

A. Quantitative determination of Ovopach by indirect ELISA

ELISA is "in house" prepared for each and every assay. The minimum detected amount of Ovopach is 10 nanograms in the tested material. Due to the specificity and reproducibility of immunoenzymatic reaction, ELISA is used in the manufacturing process of Ovopach, on production stages and during qualitative and quantitative control. a) Perform the ELISA to be used in the quantitative control of Ovopach by comparing with an international IgY standard (USA) or with an IgY substandard prepared at Romvac. b) Cover the wells with 150 μΐ anti-avian rabbit serum IgG at 3,75 μπι/ml concentration in carbonate-bicarbonate buffer;

c) Incubate the 96-ELISA well plates for 90 minutes at +37 °C;

d) Wash four times in 300 μΐ PBT-Tween using an automatic plate washer;

e) Add 200 μΐ of 1% BSA in PBS-Tween in each well and incubate for 45 minutes at 37 °C; f) Wash the reaction plate four times in PBS-Tween as per section d);

g) Add in triplicate 150 μΐ specific IgY-M or SPF IgY (25, 12,5, 6,25 μ^ηιΐ) in PBS;

h) Add in parallel in triplicate 150 μΐ IgY SIGMA standard (25, 12,5, 6,25 μΕ ηύ);

i) Incubate plates at +37°C for 90 minutes;

j) Wash plates four times in PBS-Tween;

k) Add 150 μΐ IgG anti IgY conjugated with peroxidase to 1 :5000 dilution;

1) Add 150 μΐ TMB and incubate for 5-15 minutes at room temperature in the dark m) Freeze the reaction with 150 μΐ HC1;

n) Read the reaction under D0₄₅o filter spectrophotometer;

o) Perform comparative standard curves and consider the highest positive dilution to 0,200 DO. B. Quantitative determination of Ovopach (specific IgY) by direct ELISA a) Cover the plate overnight at +4°C or for 2 hours at room temperature with IgY to be tested and standard IgY each in three replicates, in binary dilutions starting with 1 : 1000 dilution in carbonate-bicarbonate solution;

b) Keep wells Al and HI as IgY controls;

c) Wash 3 times with wash solution;

d) Add 100 μΐ anti-avian 1 :5000 diluted conjugate;

e) Incubate the plate for 2 hours at +37 °C;

f) Wash 4 times with wash solution;

g) Add 100 μΐ TMB and leave at room temperature for 5-15 min;

h) Add 100 μΐ stop solution;

i) Read the reaction absorbance under 450 nm filter spectrophotometer.

j) Assay the reaction by blank wells control where D0₄₅o should be maximum 0.060. The reaction is not valid if other values are obtained,

k) The dilution for which DO₄₅₀ is 0.200 than the international standard IgY is considered positive reaction for IgY presence.

1) Perform the μg/ml IgY content in comparison with reference IgY taking into account that

ELISA detects minimum 10 ng/ml and it is performed in comparison with standard IgY.

Both tests were carried out using IgY-PC2 anti S. aureus ROMVAC substandard. Reactions are recorded at optical intensity of 3,700 OD600 and 2,300 OD600 confirming the presence of 420 nanograms and 400 nanograms per ml of IgY-PC2.

Example 5

C. Determination of IgY contents in the blood of patients treated with IgY-specific, by ELISA

The specific anti psoriasis activity of IgY is determined by a quantitative method against the antigen represented by the inactivated and freeze-dried pathological material.

Sample: MD Serum (patient from Constanta supplemented with hyperimmune eggs + local skin treatment with personalized IgY solution) - 3 mL serum received from Romar Clinic Constanta - 250 mL serum/tube, stored at -20 °C.

Negative control: SPF IgY (26.01.2012)

Positive control: Chicken IgY, DEAE Purified - Lampire Laboratories

Conjugate IgG-anti IgY avian ICL - prepared with NaCl Sigma and MiliQ / 2015 water

Conj. diluent SurModics - StabilZyme HRP Conjugate Stabilizer

Carbonate buffer S9/2015

PBS-Tween 0,05% S16/2015.

Fixing solution PBS-Tween 20 (2%) SI 5/2015

Serum dilution buffer S4/ 2015

Coated plate washing solution S3-P /2015

Cromogen: TMB W USA

Stop solution S7 / 2014

WORKING METHOD

A. Preparation of ELISA plate

- Distribute 100 μΐ / well from the diluted suspension, as per the plate configuration;

- Allow the plate to cover for 2 hours at +37°C; After removing the liquid, wash the plate 3 times in PBS-Tween 20 (2%) wash solution.

- Freeze the reaction with fixing buffer, 300 μΐ / well and incubate for 30 minutes at room temperature;

- Remove the freezing liquid.

B. Primary reaction with the antibodies

- Distribute in each well 100 μΐ anti IgY avian conjugate diluted 1 : 10.000, using the conjugated dilution buffer as diluent.

- Incubate for 2 hours at +37 °C.

- Wash 4 times in PBS-Tween 20 (2%) wash solution.

- Add 100 μΐ TMB and leave at room temperature for 5-15 min.

- Add 100 μΐ stop solution.

- Read the absorbance at 450 nm.

The reaction has been performed accurately and the plate had no impact on the enzymatic reaction. The sample extracted from the SPF chicken egg had a negative reaction (wells CI, Dl and Dl). The positive IgY control and the IgY of the patient reacted specifically at intensity of 3,340 OD450. The immune response intensity in this experiment depends on the concentration of the antigen covering the reaction plate at 10 μg per well.

Ser pacient MD - antigen personalizat

2 3 4

Log (dilution factor)

This graphic representation proves the presence of IgY AP in blood when treatment is carried out with large amounts of IgY-specific AP. Example 6

Chronic 16-year old psoriasis treatment 16 using personalized IgY and personalized Ovopach egg- Psoriasis treatment

Patient: F/65 years old diagnosed 14 years ago with erythrodermic psoriasis. Skin wounds were found all over the body. The female patient underwent antibiotic treatment and other types of allopathic medication but with no results. After 60 days of treatment, the scalls fell and the swollen areas subsided. Itching and local aches subsided after 48-72 days of treatment. On the skin we can see the affected areas which are pink and clearly delimitate from the normal skin.

17.06.2015 after about 3 months of treatment and (B) Photo taken on 21.08.2015 after about 5 months of treatment

17.06.2015 afler about 3 months of treatment and (B) Photo taken on 21.08.2015 after about 5 months of treatment

Figure 4.^' Evolution of wounds on the legs after administering personalized IgY; (A) Photo taken on 17.06.2015 after about 3 months of treatment and (B) Photo taken on 21.08.2015 after about 5 months of treatment;

taken on 17.06.2015 after about 3 months of treatment and (B) Photo taken on 21.08.2015 after about

5 months of treatment

Example 7

Case study 4

• 45 years old M patient

6 years old Psoriasis vulgaris located on various areas

• Biological samples were taken

• Treatment was initiated with Imunoinstant poly liquid, raw egg containing IgY- polyvalent and gel with IgY-polyvalent.

• The patient undergoes treatment twice a day by washing of the wounds with soap, with IgY-poly liquid and then allowing to dry. Gel with IgY-poly is applied twice a day on the wounds.

The patient eats a raw egg in milk in the morning and performs throat wash for 2 minutes in the evening with IgY-polyvalent and then swallows the contents. After 3 days of treatment, the patient is free of itching and smarting. After 19 days of treatment the wounds start to subside, the silver-white scalls fall and the overall status of the patient is good. Photos are shown below from the beginning of treatment and after 19 days of treatment.

Figu t the beginning of treatment and (B) Photo taken after 19 days of treatment

A. B.

Figure 7: Evolution of wounds on the upper left arm after administering personalized IgY; (A) Photo taken at the beginning of treatment and (B) Photo taken after 19 days of treatment

A. B.

Figure 8: Evolution of wounds on the upper right arm after administering personalized IgY; (A) Photo taken at the beginning of treatment and (B) Photo taken after 19 days of treatment

Case study 7

65 years old patient diagnosed 14 years ago with psoriasis. The patient received whole egg, oral polyvalent immunoglobulin and local gel with IgY-polyvalent. After 30 days of treatment, the wounds stop from discharging and there is no itching and bleeding wounds. The silver-white scalls detached and the reddish areas on the skin subsided.

A. B.

Figure 9: Evolution of wounds on the skin after administering personalized IgY; (A) Photo taken at the beginning of treatment and (B) Photo taken after 30 days of treatment

gure 10: oun s on t e egs eore eginning treatment

Figure 11: Evolution of wounds on the leg after administering personalized IgY for 30 days;

33

Claims

CLAIMS Title: MANUFACTURE AND USE OF PERSONALIZED HYPERIMMUNE EGG (OVOPACH) IN PSORIASIS TREATMENT Inventors: Chiurciu Constantin, Viorica Chiurciu, Lucica Sima, luliana Mihai, Patrascu lonel Victor

1. Claims for the method of preparation of the immunogen taken from the psoriasis patient, which is used for chicken immunization;

2. Claims for the preparation of inoculums-specific immunoglobulins (specific IgY-idiotypic also called IgYl) as biological product which, in the patient's organism, transfers the information it holds on the Fab fragment, to the naive T cells which reproduce a negative cloning image. The T cells in this IgG2-type clone order the B-type cells to produce antibodies identical with the information they hold and transfer. The B-type cell reproduction of the information from the T-cell is identical with the negative of the IgY- idiotypic negative which represents a positive image, namely the IgG specific to the immunogen sampled from each and every patient. This mechanism can be explained as an immunological phenomenon in which the antigen initially sampled from the psoriasis patient is administered as immunogen in the chicken. The chicken generates an immune response and produces IgGl, namely the specific IgY- idiotypic which becomes matrix for the naive T cells. The T cells clone and transfer the IgG2-type information to the B-type cells which form a negative image of the T cell negative and produce IgG identical with specific IgY- idiotypic from chickens.

3. Claims for the hyperimmune egg from which immunoglobulins specific to each antigen existent in the sample from the psoriasis patient are extracted and from which the immunogen administered in chickens is prepared.

4. Claims for the method of treatment according to which, from the skin lesions of the psoriasis patient, a material is harvested, containing microorganisms and cell structures used for the preparation of an immunogen administered in chickens. The eggs of the chickens with serological response were used for the preparation of specific immunoglobulins as liquid (20 μg/ml of specific IgY), as spray (50 μg/ml), as gel (30 μg/ml), as cassettes with 200 μg of specific IgY /piece, as tablets with 100 and 200 μg/piece or as freeze-dried or atomized yolk 6 g/bag.

5. Claims for the method of treatment which involves immunoglobulins extracted from the yolk of chickens immunized with immunogen prepared from each and every patient, described in section 4 and the raw whole egg mixed with milk at 24-30°C temperature.